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Abstract:

The present invention relates to environmental stress-inducible 557
promoter isolated from rice, a recombinant plant expression vector
comprising said promoter, a method of producing a target protein by using
said recombinant plant expression vector, a method of producing a
transgenic plant using said recombinant plant expression vector, a
transgenic plant produced by said method, a method of improving
resistance of a plant to environmental stress by using said promoter, and
a primer set for amplification of said promoter.

Claims:

1. An environmental stress-inducible 557 promoter, the promoter
consisting of nucleotide sequence of SEQ ID NO: 1 or a nucleotide
sequence with at least 70% homology with the nucleotide sequence of SEQ
ID NO: 1.

4. The recombinant plant expression vector according to claim 3, which is
constructed by operatively linking a target gene encoding a target
protein to a downstream region of the promoter.

5. A method of producing a target protein in a plant, comprising
transforming a plant by utilizing the recombinant plant expression vector
of claim 3 to produce a transgenic plant and applying an environmental
stress to the transgenic plant to produce the target protein in the
plant.

7. A method for producing a transgenic plant comprising: transforming a
plant cell with the recombinant plant expression vector of claim 3, and
regenerating the above described transformed plant cell into a transgenic
plant.

8. A transgenic plant which is produced according to the method of claim
7.

9. The transgenic plant according to claim 8, wherein said plant is a
monocot plant.

10. A method of improving resistance of a plant to environmental stress,
comprising introducing the promoter of claim 1 to a plant.

11. The method according to claim 10, characterized in that the
environmental stress is drought stress.

Description:

CROSS REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY

[0001] The present application claims all benefits accruing under 35
U.S.C. §365(c) from the PCT International Application
PCT/KR2010/004891, with an International Filing Date of Jul. 26, 2010,
which claims the benefit of Korean patent application No. 10-2010-0029708
filed in the Korean Intellectual Property Office on April 1, 2010, the
entire contents of which are incorporated herein by reference.

BACKGROUND

[0002] 1. Technical Field

[0003] The present invention relates to environmental stress-inducible 557
promoter isolated from rice and use thereof. Specifically, the present
invention relates to environmental stress-inducible 557 promoter isolated
from rice, a recombinant plant expression vector comprising said
promoter, a method of producing a target protein by using said
recombinant plant expression vector, a method of producing a transgenic
plant using said recombinant plant expression vector, a transgenic plant
produced by said method, a method of improving resistance of a plant to
environmental stress by using said promoter, and a primer set for
amplification of said promoter.

[0004] 2. Background Art

[0005] A promoter is a part of genome which is located upstream of a
structural gene and regulates transcription of the structural gene into
mRNA. A promoter is activated by binding of various general transcription
factors, and it typically comprises a base sequence such as TATA box, CAT
box, etc. which regulates gene expression. Since proteins that are
required for basic metabolism of a living body need to be maintained at
constant concentration, a promoter that is associated with genes of such
proteins is constantly activated even by general transcription factors
only. On the other hand, for the proteins of which function is not
required during normal time but required only under specific
circumstances, an inducible promoter which can induce expression of a
corresponding structural gene is linked to the corresponding genes. In
other words, an inducible promoter is activated by binding of specific
transcription factors that are stimulated during a developmental process
of an organism or stimulated by environmental factors.

[0006] For the development of a transgenic plant based on gene
transformation, a promoter which can induce constant and strong
expression, for example cauliflower mosaic virus 35S promoter (CaMV35S,
Odell et al., Nature 313: 810-812, 1985), is widely used. However, since
constant overexpression of a specific gene linked to such promoter can
produce an excess amount of proteins that are not necessarily required
for metabolism of a normal living body, it often becomes problematic in
that a transgenic plant does not properly germinate or only a small-sized
plant is produced due to toxic effect generated by the proteins present
in an excess amount. As a representative example, it was found that
Arabidopsis thaliana in which DREB1A, a gene encoding transcription
factors responding to environmental stress, is overexpressed by using
CaMV35S promoter has improved resistance to low temperature and drought
condition, but also had a dwarfed phenotype with inhibited growth and
increased production of proline and water-soluble carbohydrates (Liu et
al., Plant Cell 10: 1391-1406, 1998; Gilmour et al., Plant Physiol. 124:
1854-1865, 2000). Occurrence of such problems can be minimized by using
an inducible promoter for rd29A, that is a gene relating to environmental
stress, instead of CaMV35S promoter (Kasuga et al, Nat. Biotechnol. 17:
287-291, 1999).

[0007] Under the circumstances, instead of a promoter which constantly
induces gene expression in a plant tissue, an inducible promoter which
can induce expression of a target gene at specific time and under
specific condition has been actively studied and developed. As a
scientific research, an inducible promoter system, which can stimulate
biosynthesis of a target protein based on introduction of a promoter
isolated from a microorganism or an animal to a plant and use of a
chemical substance as an inducing agent, has been widely developed. Until
now, as an example of application of an expression inducing system based
on use of a chemical substance on a plant, a method in which a steroid
such as dexamethasone, antibiotics such as tetracycline, and a chemical
substance such as copper ion, IPTG and the like are used as an inducing
agent has been known (Gatz et al., Plant J. 2: 397-404, 1992; Weimann et
al., Plant J. 5: 559-569, 1994; Aoyama T. and Chua N-H, Plant J. 11:
605-612, 1997). However, such method is problematic in that the chemical
substance used as an inducing agent for a system is extremely expensive
and such chemicals often have a toxic effect by themselves, so that the
method cannot be applied to all plants.

[0008] In Korean Patent Registration No. 10-0781059, an inducible promoter
that is activated by environmental stress and a method of obtaining a
transgenic protein in guard cells by using such promoter are described.
In addition, in Korean Patent Registration No. 10-0578461, a
stress-inducible promoter that is isolated from rice is disclosed.
However, these are all different from the promoter described in the
present invention.

SUMMARY

[0009] The present invention was devised in view of above-described needs.
Specifically, after intensive studies to develop a promoter which is
induced by environmental stress, particularly by drought stress,
inventors of the present invention found that a certain promoter isolated
from rice is strongly induced by drought stress, and therefore completed
the present invention.

[0010] In order to solve the above problems, the present invention
provides environmental stress-inducible 557 promoter isolated from rice.

[0012] Further, the present invention provides a method of producing a
target protein by using said recombinant plant expression vector.

[0013] Further, the present invention provides a method of producing a
transgenic plant using said recombinant plant expression vector.

[0014] Further, the present invention provides a transgenic plant produced
by said method.

[0015] Further, the present invention provides a method of improving
resistance of a plant to environmental stress by using said promoter.

[0016] Still further, the present invention provides a primer set for
amplification of said promoter.

[0017] The present invention provides an isolated nucleic acid construct
comprising a strong constitutive plant promoter which can bind to a
heterologous gene encoding a desired polypeptide in a tissue of a
transgenic plant. When used as a construct for a heterologous coding
sequence in a chimeric gene, said construct functions as a drought stress
inducible-promoter and is useful for obtaining high expression of a
desired polypeptide in a leaf tissue of a plant. A vector in which a
certain chimeric gene is linked to said construct can be introduced to a
plant tissue, and as a result, a plant can be deliberately transformed
with such vector and a foreign material can be produced from the
resulting transgenic plant.

[0018] In addition, it is expected that, by using the promoter that is
provided by the present invention and a technology of breeding an
environmental stress-resistant transgenic plant using the promoter,
improvement of crop yield for the economically important crops can be
achieved.

[0023] In order to achieve the object of the invention described above,
the present invention provides environmental stress-inducible 557
promoter isolated from rice, which includes a nucleotide sequence of SEQ
ID NO: 1.

[0024] The present invention is related to a specific promoter that is
isolated from rice, and more specifically, the promoter includes a
nucleotide sequence of SEQ ID NO: 1. Further, variants of the said
promoter sequence are within the scope of the present invention. The
variants have a different nucleotide sequence but have similar functional
characteristics to those of the nucleotide sequence of SEQ ID NO: 1.
Specifically, the promoter of the present invention may comprise a
nucleotide sequence with at least 70%, preferably at least 80%, more
preferably at least 90%, and most preferably at least 95% homology with
the nucleotide sequence of SEQ ID NO: 1.

[0025] The "sequence homology %" for a certain polynucleotide is
identified by comparing a comparative region with two sequences that are
optimally aligned. In this regard, a part of the polynucleotide in
comparative region may comprise an addition or a deletion (i.e., a gap)
compared to a reference sequence (without any addition or deletion)
relative to the optimized alignment of the two sequences.

[0026] With respect to the promoter according to one embodiment of the
present invention, the environmental stress can be drought, high
temperature, cold temperature, high salt, heavy metals, etc. For example,
it is drought stress.

[0027] In order to achieve another object of the present invention, an
embodiment of the present invention provides a recombinant plant
expression vector which comprises the promoter according to the present
invention. As an example of the recombinant plant expression vector,
pHC10::557promoter vector shown in FIG. 2 of the present invention can be
mentioned, but not limited thereto.

[0028] The term "recombinant" indicates a cell which replicates a
heterogeneous nucleotide or expresses the nucleotide, a peptide, a
heterogeneous peptide, or a protein encoded by a heterogeneous
nucleotide. Recombinant cell can express a gene or a gene fragment, that
are not found in natural state of cell, in a form of a sense or
antisense. In addition, a recombinant cell can express a gene that is
found in natural state, provided that said gene is modified and
re-introduced into the cell by an artificial means.

[0029] The term "vector" is used herein to refer DNA fragment (s) and
nucleotide molecules that are delivered to a cell. Vector can be used for
the replication of DNA and be independently reproduced in a host cell.
The terms "delivery system" and "vector" are often interchangeably used.
The term "expression vector" means a recombinant DNA molecule comprising
a desired coding sequence and other appropriate nucleotide sequences that
are essential for the expression of the operatively-linked coding
sequence in a specific host organism. Promoter, enhancer, termination
codon and polyadenylation signal that can be used for an eukaryotic cell
are well known in the pertinent art.

[0030] A preferred example of plant expression vector is Ti-plasmid vector
which can transfer a part of itself, i.e., so called T-region, to a plant
cell when the vector is present in an appropriate host such as
Agrobacterium tumefaciens. Other types of Ti-plasmid vector (see, EP 0
116 718 B1) are currently used for transferring a hybrid gene to
protoplasts that can produce a new plant by appropriately inserting a
plant cell or hybrid DNA to a genome of a plant. Especially preferred
form of Ti-plasmid vector is a so called binary vector which has been
disclosed in EP 0 120 516 B1 and U.S. Pat. No. 4,940,838. Other vector
that can be used for introducing the DNA of the present invention to a
host plant can be selected from a double-stranded plant virus (e.g.,
CaMV), a single-stranded plant virus, and a viral vector which can be
originated from Gemini virus, etc., for example a non-complete plant
viral vector. Use of said vector can be advantageous especially when a
plant host cannot be appropriately transformed.

[0031] Expression vector would comprise at least one selective marker.
Said selective marker is a nucleotide sequence having a property based on
that it can be selected by a common chemical method. Every gene which can
be used for the differentiation of transformed cells from non-transformed
cell can be a selective marker. Example includes, a gene resistant to
herbicide such as glyphosate and phosphintricin, and a gene resistant to
antibiotics such as kanamycin, G418, bleomycin, hygromycin, and
chloramphenicol, but not limited thereto.

[0032] For the plant expression vector according to one embodiment of the
present invention, any conventional terminator can be used. Example
includes, nopaline synthase (NOS), rice a-amylase RAmyl A terminator,
phaseoline terminator, and a terminator for optopine gene of
Agrobacterium tumefaciens, etc., but are not limited thereto. Regarding
the necessity of terminator, it is generally known that such region can
increase a reliability and an efficiency of transcription in plant cells.
Therefore, the use of terminator is highly preferable in view of the
contexts of the present invention.

[0033] With respect to the recombinant plant expression vector according
to one embodiment of the present invention, it can be the one which is
constructed by operatively linking a gene encoding a target protein to a
downstream region of the promoter of the present invention. In the
present specification, the term "operatively-linked" is related to a
component of an expression cassette, which functions as a unit for
expressing a heterogenous protein. For example, a promoter which is
operatively-linked to a heterogeneous DNA encoding a protein stimulates
production of functional mRNA which corresponds to the heterogeneous DNA.

[0034] The above-described target protein can be any kind of protein, and
examples thereof include a protein which is therapeutically useful, i.e.,
an enzyme, a hormone, an antibody, a cytokine, etc., a protein which can
accumulate a great amount of nutritional components useful for health
enhancement in an animal including a human, and an enzyme which can
degrade cellulose, etc., but not limited thereto. Specific examples of a
target protein include interleukin, interferon, platelet-derived growth
factor, hemoglobin, elastin, collagen, insulin, fibroblast growth factor,
human growth factor, human serum albumin, erythropoietin,
cellobiohydrolase, endocellulase, beta-gluosidase, xylanase and the like.

[0035] In addition, the present invention provides a method of producing a
target protein in a plant characterized in that a plant is transformed
with the above-described recombinant plant expression vector, and
environmental stress, preferably drought stress, is applied to a
resulting transgenic plant to produce a target protein in the plant. The
target protein which can be obtained according to this method is the same
as those described above.

[0036] Plant transformation means any method by which DNA is delivered to
a plant. Such transformation method does not necessarily need a period
for regeneration and/or tissue culture. Transformation of plant species
is now quite general not only for dicot plants but also for monocot
plants. In principle, any transformation method can be used for
introducing a hybrid DNA of the present invention to appropriate
progenitor cells. The method can be appropriately selected from a
calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al.,
1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol.
8, 363-373), an electroporation method for protoplasts (Shillito R. D. et
al., 1985 Bio/Technol. 3, 1099-1102), a microscopic injection method for
plant components (Crossway A. et al., 1986, Mol. Gen. Genet. 202,
179-185), a particle bombardment method for various plant components (DNA
or RNA-coated) (Klein T. M. et al., 1987, Nature 327, 70), or a
(non-complete) viral infection method in Agrobacterium tumefaciens
mediated gene transfer by plant invasion or transformation of fully
ripened pollen or microspore (EP 0 301 316), etc. A method preferred in
the present invention includes Agrobacterium mediated DNA transfer. In
particular, so-called binary vector technique as disclosed in EP A 120
516 and U.S. Pat. No. 4,940,838 can be preferably adopted for the present
invention.

[0037] The "plant cell" that can be used for the plant transformation in
the present invention can be any type of plant cell. It includes a
cultured cell, a cultured tissue, a cultured organ or a whole plant.
Preferably, it is a cultured cell, a cultured tissue, or a cultured
organ. More preferably, it is a cultured cell in any form.

[0038] The term "plant tissue" can be either differentiated or
undifferentiated plant tissue, including root, stem, leaf, pollen, seed,
cancerous tissue and cells having various shape that are used for
culture, i.e., single cell, protoplast, bud and callus tissue, but not
limited thereto. Plant tissue can be in planta or in a state of organ
culture, tissue culture or cell culture.

[0040] transforming a plant cell with the recombinant plant expression
vector of an embodiment of the present invention, and

[0041] regenerating the above described transformed plant cell into a
transgenic plant.

[0042] The method of the present invention comprises a step of
transforming a plant cell with the recombinant vector of an embodiment of
the present invention, and such transformation may be mediated by
Agrobacterium tumefaciens. In addition, the method of the present
invention comprises a step of regenerating a transformed plant cell to a
transgenic plant. A method of regenerating a transformed plant cell to a
transgenic plant can be any method that is well known in the pertinent
art.

[0044] Further, the present invention provides a method of improving
resistance of a plant to environmental stress by introducing the promoter
of an embodiment of the present invention to a plant. The environmental
stress can be preferably drought stress, but not limited thereto. This
method can be carried out by operatively linking a structural gene which
can improve resistance to stress to the promoter of the present invention
and then introducing the resultant gene to a plant. The structural gene
which can improve resistance to stress encodes a protein which is
responsible for improving resistance of a plant to environmental stress
such as dehydration, low temperature, or salt stress, etc. Examples of
such protein are as follows: LEA protein; water channel protein; a
synthetic enzyme for a common solute; tobacco detoxifying enzyme; an
enzyme for synthesis of a substance which regulates osmotic pressure
(e.g., sucrose, proline or glycine betaine); a gene which encodes omega-3
fatty acid desaturase of Arabidopsis thaliana, which is an enzyme for
altering composition of a cell membrane and a gene which encodes
D9-desaturase of cyanobacteria; P5CS, which is a main enzyme for proline
synthesis, and; AtGo1S3 gene for galactinol synthesis.

[0045] Still further, the present invention provides a primer set for
amplification of the promoter of the present invention, including a
nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3.

[0046] The primer can be an oligonucleotide which including a fragment of
at least 16, at least 17, at least 18, at least 19, at least 20, at least
21, at least 22, at least 23, at least 24, and more than 25 consecutive
nucleotides of SEQ ID NO: 2.

[0047] The primer can be an oligonucleotide which including a fragment of
at least 16, at least 17, at least 18, at least 19, at least 20, at least
21, at least 22, at least 23, at least 24, and more than 25 consecutive
nucleotides of SEQ ID NO: 3.

[0048] In the present specification, the term "primer" indicates a
single-stranded oligonucleotide sequence which is complementary to a
nucleic acid strand to be copied, and it can be a starting point for
synthesis of a primer extension product. Length and sequence of a primer
are selected so as to allow the synthesis of a primer extension product.
Specific length and sequence of a primer are determined in view of primer
use condition such as temperature, ionic strength and the like, as well
as complexity of a target DNA or RNA.

[0049] According to the present invention, an oligonucleotide which is
used as a primer may also comprise a nucleotide analogue such as
phosphorothioate, alkyl phosphorothioate or peptide nucleic acid, and
also an intercalating agent.

[0050] The present invention will now be described in greater detail with
reference to the following examples. However, it is only to specifically
exemplify the present invention and in no case the scope of the present
invention is limited by these examples.

EXAMPLES

Example 1

Isolation of Genomic DNA from Whole Rice Plant

[0051] Leaves of an about three-week old rice plant (Dong-jin variety,
Oryza sativa L., 1 gram) were harvested and frozen in liquid nitrogen,
and then by using a pulverizer leaf powder was prepared. With a DNeasy
Plant mini kit (Qiagen), 100 g of the leaf powder was added with 400 ul
AP1 and 4 ul RNaseA. The mixture was kept at 65° C. for 10
minutes. After adding 130 ul of AP2, the mixture was kept on ice for 5
minutes followed by centrifuge at 14,000 rpm for 2 minutes. Then, to
remove polysaccharides, the supernatant was introduced to a spin column
in divided portions (650 ul each) and centrifuged. The aqueous solution
was collected and added with AP3/E which is 1.5-fold the amount of the
solution. The mixture was added to a DNA binding column in divided
portions (650 ul each) and centrifuged at 8000 rpm for 1 minute. After
washing twice with AW buffer (500 ul), the column was dried well and then
dissolved in sterilized water (100 ul).

Example 2

PCR Cloning of 1789bp 557 Gene Promoter

[0052] For cloning 557 promoter, a gene-specific primer set of
557pro5'-HindIII: 5'-AAGCTTGTCGGTTGTTGCCTTTTCAT-3' (SEQ ID NO: 2) and
557pro3'-PstI: 5'-CTGCAGGCTCGCGATCAAGAAAGAAG-3' (SEQ ID NO: 3) was used
and the genomic DNA was amplified by polymerase chain reaction (PCR). PCR
reaction solution comprised DNA 200 ng, 10×ExTaq polymerase buffer
solution (pH 8.0), 200 mM dNTPs, 5×BandDoctor, 10 pmole of each
primer and 5U ExTaq polymerase (Takara Bio.). The reaction was started
without adding any enzyme, and then amplification was carried out
according to the following condition: 1 cycle including heating at
95° C. for 5 minutes as pre-denaturation step; 35 cycles including
heating at 95° C. for 1 minute, 64° C. for 1 minute and
72° C. for 2 minutes, and; 1 cycle including heating at 72°
C. for 10 minutes as a post-extension in GeneAmp PCR system 9700
amplifier (Applied Biosystem). Major products that had been obtained from
the amplification by PCR was gel-purified by using Gel Purification Kit
(Bioneer, Korea). After subsequent cloning into pGEM-T easy vector
(Promega, USA), the plasmid construct pGEMTvec557promoter (FIG. 1) was
produced according to the manufacturer's instruction, and then sequenced
(SEQ ID NO: 1).

Example 3

Construction of a Vector for 557 Promoter Expression

[0053] DNA fusion construct for the promoter which comprises a reporter
gene encoding mGFP was constructed as follows. For easy cloning into a
binary vector pHC10, plasmid pGEMTvec557promoter was digested with PstI
and Sall, and then subcloned into PstI and Sall sites of pHC10. These
clones were named pHC10::557promoter, and an expression vector for
delivery of plant transformation was produced (FIG. 2).

Example 4

Evaluation of the Function of 557 Promoter Gene Under Drought Stress
(RT-PCR Analysis)

[0054] Three-week old rice leaves were dried (for Ohr, 6 hr, 12 hr or 24
hr) and after pulverizing them with liquid nitrogen freezing, 100 mg of
thus-obtained powder was added with TRI reagent (1 ml), and left at room
temperature for 5 minutes. Bromochloropropane (0.1 ml) was added,
followed by maintaining the mixture at room temperature for 15 minutes
and centrifuge at 4° C., 12000 rpm for 15 minutes. Only the
supernatant was transferred to a new tube, added with isopropanol
(0.65-fold amount) and the mixture was kept at room temperature for 10
minutes. After centrifuge at 4° C., 12000 rpm for 15 minutes, RNA
pellet was washed with 75% DEPC-ethanol, dried and dissolved in 30 ul
solution. Gel-electrophoresis was carried out (1% agarose-formaldehyde
gel) for identification, and by using 5 ug of the total RNA, 1st
cDNA was synthesized according to the following condition: total RNA 5
ug, Oligo dT (10pmole), and 10 mM dNTPs were added and the mixture was
heated at 65° C. for 5 minutes; 5×First strand buffer, 0.1M
DTT, and RNaseOut (40 U/ul) were added and the mixture was heated at
42° C. for 2 minutes and; SuperScript® II (200 units) was added
and the mixture was heated at 42° C. for 50 minutes and further at
72° C. for 15 minutes. The resultant was used for PCR
determination. By using a primer set of 557 GSP-F:
5'-CCAGTTGCGTATGACATCGCT-3' (SEQ ID NO: 4) and 557 GSP-R:
5'-TCTTTCTTCTTTTCACAGGGAG-3' (SEQ ID NO: 5), PCR amplification was
carried out according to the following condition: 1 cycle including
heating at 95° C. for 5 minutes as pre-denaturation step; 25
cycles including heating at 95° C. for 30 seconds, 58° C.
for 30 seconds and 72° C. for 30 seconds, and; 1 cycle including
heating at 72° C. for 5 minutes as a post-extension in GeneAmp PCR
system 9700 amplifier (Applied Biosystem). Major products that had been
amplified by PCR were confirmed by gel -electrophoresis (2.5% agarose
gel; see FIG. 3).

[0055] As it is shown in FIG. 3, compared to the rice leaves which did not
receive any drought stress, gene expression was increased in the rice
leaves which received drought stress. Thus, it was found that the
promoter of the present invention is a drought stress-inducible promoter.

Example 5

GFP Determination After Transformation into Rice Callus

[0056] pHC10::557promoter vector was introduced to the rice plant
(Dong-jin variety, Oryza sativa L.) based on Agrobacterium-mediated
vacuum infiltration method. To select a hygromycin resistant gene
variant, unpolished rice in which testa was removed for callus formation
was used as rice seeds. For the removal of testa, a simple rice polishing
machine was used and the rice seeds with removed testa were sterilized
once with 70% ethanol for 10 minutes, followed by 30 minutes
sterilization in 2 to 3% sodium hypochlorite solution and washing with
sterilized water at least three times. The rice seeds obtained after the
sterilization were placed on a medium for callus formation (i.e., 10 to
12 seeds per medium) and incubated under dark condition at 28° C.
for 3 to 4 weeks. Then, light yellow callus (1 to 2 mm diameter) was
selected and cultured in 2N6 medium for three days. Meanwhile, the day
after the callus selection, Agrobacterium was added to a medium
comprising carbenicillin and hygromycin and cultured under dark condition
at 28° C. for 3 to 5 days. After transferring the callus which had
been cultured for three days, it was co-cultured in suspension comprising
Agrobacterium and further cultured under dark condition at 25° C.
for 3 days. The resulting co-cultured callus was washed with sterilized
water comprising carbenicillin and the transformed callus was cultured
under dark condition at 28° C. for 2 to 3 weeks. Healthy callus
showing good growth was selected again. After the transfer to a new
medium, the callus was again cultured under the same condition. Healthy
callus was then again added to a regeneration selection medium. The
transformant in which the target gene had been introduced was identified
by genomic PCR. [0057] GFP Determination

[0058] In order to examine the function of 557 promoter, an enzyme-linked
immunosorbent assay (ELISA) analysis using an GFP antibody was carried
out. Specifically, after applying Ohr or 6 hr drought stress to the
transformant leaf which had been selected by genomic PCR, total protein
was extracted by using a solution comprising 50 mM Tris-HCl (pH 7.5), 1
mM EDTA, 8 mM MgCl2, and a proteinase inhibitor. Thus-obtained total
protein was quantified by using Bradford assay solution, and bug of the
protein was used for the determination. In a microplate, the antigen
comprising 200 ul coating buffer and the total protein was added and
reacted at 4° C. for 12 hours. After blocking with 1×PBS
comprising 4% skim milk for two hours at room temperature, GFP antibody
was diluted to 1/4000 with 1×PBS comprising 1% skim milk and was
allowed to bind to the antigen by incubating them at room temperature for
2 hours. 1×PBS comprising 0.05% Tween 20 was used for washing, and
the secondary antibody was diluted to 1/2000 with 1×PBS comprising
1% skim milk and was allowed to bind to the antigen by incubating them at
room temperature for 1 hour followed by washing. After the development
with a solution comprising 30% H2O2, PC buffer and OPD, the
reaction was terminated by adding 2.5M H2SO4. Then, the
absorbance at 490 nm was determined by using a microplate reader and the
result was analyzed (FIG. 4).

[0059] As it is shown in FIG. 4, compared to the transgenic rice which did
not receive any drought stress, GFP expression was significantly
increased in the transgenic rice which received drought stress. Thus, it
was found that the promoter of the present invention is a drought
stress-inducible promoter.